The present invention relates to a modification of an immunoassay based on complement-dependent immune lysis of microcapsules and liquid reagents used therefor which have been stabilized and can be used in autoanalyzers.
Immunoassay is a measuring method utilizing antigen-antibody reaction and is widely used, for example, as a method for measuring trace components in body fluids specifically.
As immunoassays which are now generally often used, there may be exemplified radioimmunoassay (RIA), enzyme immunoassay (EIA), etc. These method permit quantitative measurement of trace components in samples but involve individual problems. That is, RIA is disadvantageous, for example, in that since isotopes should be used therein, RIA requires special facilities and troublesome disposal of wastes. EIA is disadvantageous, for example, in that it requires a relatively long measurement time and is difficult to apply to an autoanalyzer.
Therefore, as an immunoassay involving none of these problems, there has recently been proposed and noted an immunoassay based on complement-dependent immune lysis of microcapsules.
A typical example of this method is a method using microcapsules (e.g. lipid film vehicles (liposomes)) having an analyte to be measured on surfaces thereof and containing a marker (e.g. enzyme) therein (hereinafter abbreviated as "labeled microcapsules") (hereinafter abbreviated as "immunoassay method based on complement-dependent immune lysis of microcapsules") (Japanese Patent Appln. Kokai No. 56-132564, U.S. Pat. No. 4,342,826). This method is as follows. A sample, labeled microcapsules, and an antibody to an analyte to be measured are fixed to carry out the antigen-antibody reaction, after which complement is added. The complement is activated by an antigen-antibody complex formed on the surfaces of the labeled microcapsules and lyses the membrane walls of the microcapsules to liberate a marker from the labeled microcapsules. The amount of analyte to be measured in the sample is determined on the basis of the amount of the marker liberated. This immunoassay method based on complement-dependent immune lysis of microcapsules induced by antigen-antibody reaction and hence makes it possible not only to measure a slight amount of an analyte to be measured, specifically, but also to carry out a series of reactions in a uniform reaction system. Therefore, it permits simpler and more rapid measurement than do conventional methods such as RIA and EIA.
This method is, however, difficult to apply to an autoanalyzer, in particular, an autoanalyzer using two liquid reagents which is now a leading autoanalyzer, and substantially no case has been reported for application of the method to such an autoanalyzer. The cause of this is that when the reagents (labeled microcapsules, antibody to an analyte to be measured, and complement) used in said method are stored in a proper combination in two liquid reagents, the stability of the liquid reagents is not sufficient.
When the three main reagents used in said method are divided between and stored in two liquid reagents, the following three combinations are thought of:
a) a combination of a mixed solution of complement and labeled microcapsules, and an antibody solution,
b) a combination of a mixed solution of complement and antibody, and a solution of labeled microcapsules,
c) a combination of a mixed solution of labeled microcapsules, and an antibody, and complement solution.
Whichever combination among them is employed for preparing liquid reagents, the resulting liquid reagents have a low stability during storage and hence are of no practical use. For example, in the case of the combination of a), the complement is inactivated by the interaction between the labeled microcapsules and the complement which coexist in the liquid reagent, so that immunolysis is gradually reduced. In the case of the combination of b), when an analyte to be measured is a substance which is unlikely to get mixed in a solution of complement derived from an animal, for example, a drug, the resulting liquid reagent is stable to a certain extent. But when it is a substance which is very likely to exist in a solution of complement, for example, hormone or tumor marker, the antigen-antibody reaction and inactivation of the complement occur in the mixed solution of the complement and the antibody, so that the stability of this liquid reagent is lowered. In the case of the combination of c), an analyte to be measured (an antigen) fixed on the labeled microcapsules reacts with the antibody previously, resulting in lowering of the measurement sensitivity. Therefore, depending on an analyte to be measured, there is now often no choice but to use three liquid reagents for an immunoassay method based on complement-dependent immune lysis of microcapsules. Accordingly, there has been an eager desire for the advent of liquid reagents for immunoassay method based on complement-dependent immune lysis of microcapsules and using two liquid reagents which can be used in autoanalyzers.